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Vlastnosti DNA vazebných mutant proteinů CSL
Teska, Mikoláš ; Folk, Petr (advisor) ; Šťovíček, Vratislav (referee)
Notch pathway plays a critical role during the development and life of metazoan organisms. CSL proteins are the component of the Notch pathway that mediates the regulation of target genes. The discovery of CSL-like proteins in yeast raised the question of their function in unicellular organisms which did not utilize the canonical Notch pathway. CSL-homologues in yeast are conserved in parts that are important for DNA binding and for fission yeast proteins it was shown that they bind to CSL recognition elements in vitro. In fission yeast, CSL paralogues Cbf11 and Cbf12 play antagonistic roles in cell adhesion and the coordination of cell and nuclear division. Yeast CSL proteins have long and intrinsically unstructured N- terminal domains compared to metazoan CSL proteins. In this study, we investigated the functional significance of these extended N-termini of CSL proteins by their complete removal. For newly constructed truncated variants of proteins Cbf11 and Cbf12 in Schizosaccharomyces pombe we observed the lack of ability to bind CSL recognition RBP probe. The removal of N-terminal parts of CSL proteins in fission yeast led to the change in their cellular localization. Once strongly preferred nuclear localization changed by the removal of N-terminal domains to cytoplasmic localization with a...
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Vlastnosti DNA vazebných mutant proteinů CSL
Teska, Mikoláš ; Folk, Petr (advisor) ; Šťovíček, Vratislav (referee)
Notch pathway plays a critical role during the development and life of metazoan organisms. CSL proteins are the component of the Notch pathway that mediates the regulation of target genes. The discovery of CSL-like proteins in yeast raised the question of their function in unicellular organisms which did not utilize the canonical Notch pathway. CSL-homologues in yeast are conserved in parts that are important for DNA binding and for fission yeast proteins it was shown that they bind to CSL recognition elements in vitro. In fission yeast, CSL paralogues Cbf11 and Cbf12 play antagonistic roles in cell adhesion and the coordination of cell and nuclear division. Yeast CSL proteins have long and intrinsically unstructured N- terminal domains compared to metazoan CSL proteins. In this study, we investigated the functional significance of these extended N-termini of CSL proteins by their complete removal. For newly constructed truncated variants of proteins Cbf11 and Cbf12 in Schizosaccharomyces pombe we observed the lack of ability to bind CSL recognition RBP probe. The removal of N-terminal parts of CSL proteins in fission yeast led to the change in their cellular localization. Once strongly preferred nuclear localization changed by the removal of N-terminal domains to cytoplasmic localization with a...
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Notch-independent functions of CSL transcription factors
Teska, Mikoláš ; Folk, Petr (advisor) ; Převorovský, Martin (referee) ; Kuthan, Martin (referee)
Notch pathway plays a critical role during development and life of Metazoan organisms. CBF1 is a component of the Notch pathway that mediates the regulation of target genes. The discovery of CBF1-like proteins in yeast raised the question of their function in unicellular organisms - before the origin of canonical Notch pathway. CBF1-homologs in yeast are conserved in parts that are important for DNA binding and bind to CBF1-binding elements in vitro. CBF1 and related transcription factors in Metazoa (CSL) interact with many proteins in Notch-dependent as well as Notch-independent complexes. The Notch receptor has likewise some CSL-independent functions. This assay reports about interacting partners of CSL in Metazoa along with homologous proteins in yeast with the aim to highlight potential interactions of CBF1-homologs in evolutionary ancestral context.
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DNA-binding properties of the CSL proteins of Schizosaccharomyces pombe
Ptáčková, Martina ; Půta, František (advisor) ; Doležal, Pavel (referee)
As the effector component of the Notch signaling pathway the transcription factors of the CSL family (CBF1/RBP-Jκ/Suppressor of Hairless/Lag-1) are essential for many developmental processes in metazoan organisms, but they can function also independently of Notch. Recently, their presence was proved in fungal organisms lacking the Notch pathway as well as most of the known metazoan interacting partners. Cbf11 and Cbf12, the CSL proteins of the unicellular yeast Schizosaccharomyces pombe, were determined experimentally as non-essential nuclear transcription factors, which regulate cell adhesion, extracellular material production, colony morphology, septation and daughter cell separation, coordination of nuclear and cell division, and ploidy maintenance in an antagonistic way. The responsive genes of these factors are not known yet. In this study, genes of S. pombe, whose promoter regions represent potential direct targets for the Cbf proteins binding, were predicted. The binding of the Cbf11 and Cbf12 proteins, and of a truncated version Cbf12∆N to CSL response elements contained in the regulatory regions of selected S. pombe genes was tested in vitro by EMSA, and consequently, in the case of the Cbf11 protein, also in vivo by ChIP. Cbf11 and Cbf12∆N recognize specifically the response elements in...
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